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Nuclear Medicine and Biology 39 (2012) 215–226www.elsevier.com/locate/nucmedbio

Usefulness of [18F]-DA and [18F]-DOPA for PET imaging in a mousemodel of pheochromocytoma

Lucia Martiniovaa, j, Susannah Clearyb, Edwin W. Laia, Dale O. Kiesewetterc, Jurgen Seideld,Linda F. Dawsone, Jacqueline K. Phillipse, f, David Thomassong, Xiaoyuan Chenc,

Graeme Eisenhoferh, James F. Powers i, Richard Kvetnanskyj, Karel Pacaka,⁎aProgram in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH,

Bethesda, MD 20892, USAbClinical Neurocardiology Section, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892, USA

cLaboratory for Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, MD 20892, USAdMolecular Imaging Program, National Cancer Institute, NIH, Bethesda, MD 20892, USA

eFaculty of Health Sciences, Murdoch University, South St. Murdoch, Perth, WA 6150, AustraliafAustralian School of Advanced Medicine, Macquarie University, Sydney, NSW 2109, Australia

gLaboratory of Diagnostic Radiology, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USAhInstitute of Clinical Chemistry and Laboratory Medicine and the Department of Medicine, University of Dresden, Dresden, 01307 Germany

iDepartment of Pathology, Tufts Medical Center, Boston, MA 02111, USAjInstitute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, 83306 Slovakia

Received 7 February 2011; received in revised form 20 July 2011; accepted 26 July 2011

Abstract

Purpose: To evaluate the usefulness of [18F]-6-fluorodopamine ([18F]-DA) and [18F]-L-6-fluoro-3,4-dihydroxyphenylalanine ([18F]-DOPA)positron emission tomography (PET) in the detection of subcutaneous (s.c.) and metastatic pheochromocytoma in mice; to assess theexpression of the norepinephrine transporter (NET) and vesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2), all important for[18F]-DA and [18F]-DOPA uptake. Furthermore, to compare tumor detection by micro-computed tomography (microCT) to magneticresonance imaging (MRI) in individual mouse.Methods: SUVmax values were calculated from [18F]-DA and [18F]-DOPA PET, tumor-to-liver ratios (TLR) were obtained and expressionof NET, VMAT1 and VMAT2 was evaluated.Results: [18F]-DA detected less metastatic lesions compared to [18F]-DOPA. TLR values for liver metastases were 2.26–2.71 for [18F]-DOPA and 1.83–2.83 for [18F]-DA. A limited uptake of [18F]-DA was found in s.c. tumors (TLR=0.22-0.27) compared to [18F]-DOPA(TLR=1.56-2.24). Overall, NET and VMAT2 were expressed in all organ and s.c. tumors. However, s.c. tumors lacked expression ofVMAT1. We confirmed [18F]-DA's high affinity for the NET for its uptake and VMAT1 and VMAT2 for its storage and retention inpheochromocytoma cell vesicles. In contrast, [18F]-DOPA was found to utilize only VMAT2.Conclusion: MRI was superior in the detection of all organ tumors compared to microCT and PET. [18F]-DOPA had overall bettersensitivity than [18F]-DA for the detection of metastases. Subcutaneous tumors were localized only with [18F]-DOPA, a finding that mayreflect differences in expression of VMAT1 and VMAT2, perhaps similar to some patients with pheochromocytoma where [18F]-DOPAprovides better visualization of lesions than [18F]-DA.© 2012 Elsevier Inc. All rights reserved.

Abbreviations: PET, positron emission tomography; MPC cells, mouse pheochromocytoma cells; ROI, region of interest; FOV, field of view; TBR, tumor-to-background ratio; TLR, tumor-to-liver ratio; SUVmax, the maximum standardized uptake value; 2D-OSEM, 2-dimensional ordered-subsets expectationmaximization; FWHM, full-width at half-maximum; [18F]-DA, [18F]-6-fluorodopamine; [18F]-DOPA, [18F]-L-6-fluoro-3,4-dihydroxyphenylalanine; VMAT1and VMAT2, vesicular monoamine transporters 1 and 2; NET, membrane norepinephrine transporter; TH, tyrosine hydroxylase; MRI, magnetic resonanceimaging; s.c., subcutaneous; i.v., intravenous.

⁎ Corresponding author. NICHD, NIH, 10 Center Drive MSC-1109, Bethesda, MD, 20892-1109 USA. Tel.: +1 301 402 4594; fax: +1 301 402 4712.E-mail address: karel@mail.nih.gov (K. Pacak).

0969-8051/$ – see front matter © 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.nucmedbio.2011.07.007

216 L. Martiniova et al. / Nuclear Medicine and Biology 39 (2012) 215–226

Keywords: Pheochromocytoma; PET; MicroCT; MRI; Metastatic mice model; Fluorodopamine; Fluorodopa; Norepinephrine transporter; Vesicularmonoamine transporter

1. Introduction

In clinical practice, various imaging techniques are usedto localize pheochromocytoma, a rare neuroendocrine tumorarising from chromaffin cells [1]. These tumors arecharacterized by the synthesis, storage, metabolism andrelease of catecholamines [2]. Even though metastaticpheochromocytoma is generally slow growing, the prognosisof patients with disseminated disease is often poor, with a 5-year survival rate of usually less than 50% [3,4]. This is dueto the fact that, currently, there is no effective chemother-apeutic regimen [5]. Therefore, the development of animalmodels for testing new imaging methods and probes tovisualize and monitor tumor growth, as well as other tumorcharacteristics, is crucial for new drug development.

In recent years, positron emission tomography (PET) hasrapidly become a valuable diagnostic tool in the study ofpheochromocytomas, particularly with the development ofnew targeted PET radiopharmaceuticals such as [18F]-6-fluorodopamine ([18F]-DA) [6,7] and [18F]-L-6-fluoro-3,4-dihydroxyphenylalanine ([18F]-DOPA) [8]. Functional im-aging studies with [18F]-DA and [18F]-DOPA have theadvantage over anatomical imaging of superior specificityfor identification of pheochromocytomas [9,10]. Uptake of[18F]-DA, a dopamine analogue, occurs via the cellmembrane norepinephrine transporter (NET) [11]. Desipra-mine, a tricyclic anti-depressant, functions as an inhibitor ofthe uptake-1 mechanism involved in amine transport intocells [12] and has been used to evaluate the specificity of theNET transporter for [18F]-DA uptake [13,14]. [18F]-DOPA,an analogue of the dopamine precursor, DOPA, is incorpo-rated into tumor cells via the aromatic amino acid transporter[15]. Upon entering the cells, [18F]-DOPA is decarboxylatedby the aromatic amino acid decarboxylase to [18F]-DA andsequestered by vesicular monoamine transporters (VMAT1and VMAT2) into catecholamine storage vesicles. Thearomatic amino acid transporters, NET, VMAT1 andVMAT2 are specifically expressed in chromaffin cells andpheochromocytomas [16]. In this study, VMAT inhibitors,such as reserpine (VMAT1/VMAT2 inhibitor) and tetra-benazine (VMAT2 inhibitor) [17], have been used toevaluate the importance of [18F]-DA and [18F]-DOPAuptake in mouse pheochromocytoma (MPC) cells.

Recently, we have described an animal model ofmetastatic pheochromocytoma and introduced [18F]-DAPET for the localization of liver lesions [18]. The aims ofthe present study were to compare the feasibility of apotentially more practical subcutaneous (s.c.) pheochromo-cytoma model against our previously established metastaticmodel, to evaluate [18F]-DOPA PET as an alternativemodality for localization of lesions compared to micro-

computed tomography (microCT) and magnetic resonanceimaging (MRI) and to assess the importance of NET andVMATs in entry and storage of [18F]-DOPA and [18F]-DAin cells and tumors. These findings were aimed to determinewhether these pheochromocytoma models are comparable toclinical imaging scenarios and, thus, appropriate for potentialexperiment drug treatments. The metastatic model involveddiffuse spread of tumors after tail vein injection of MPC cellsand the s.c. model represented by s.c. tumors after s.c.implantation of MPC cells.

2. Materials and methods

2.1. Cell culture and in vitro assays of [18F]-DA and[18F]-DOPA uptake and storage

MPC cells were kindly provided by Dr. A.S. Tischler [19]and were maintained in Dulbecco's Modified MinimumEssential Medium (GIBCO, Grand Island, NY, USA)supplemented with 10% (v/v) fetal calf serum, 5% fetalbovine serum and 1% (v/v) penicillin/streptomycin andmaintained at 37°C and 5% CO2.

To confirm the ability of MPC cells to incorporate[18F]-DA or [18F]-DOPA, in vitro radionuclide assays wereconducted. MPC cells were seeded onto collagen-coated12-well plates at a density of 2×105 cells per well andincubated for 24 h. At the commencement of uptake assays,cells were washed three times with phosphate-bufferedsaline (PBS, pH 7.4). Cells were then incubated for 30 minwith the non-selective monoamine transporter antagonists:desipramine (1 μM, Sigma Chemical, St. Louis, MO,USA); tetrabenazine (10 μM, Sigma Chemical), aninhibitor of VMAT 2 [20,21] and reserpine (10 μM,Serpasil, Siba, Summit, NJ) an inhibitor of both VMAT1and VMAT2 [22]. Uptake studies involved incubation ofcells with 0.024 MBq/ml of [18F]-DA or 0.029 MBq/ml of[18F]-DOPA. Cells were harvested after incubation periodsof either 10 (to assess “uptake”) or 120 min (to assess“storage and retention”).

At the termination of the radionuclide assays, cells werecounted, and the radioactivity was measured on a Packardgamma counter (United Technologies). The uptake of [18F]-DA and [18F]-DOPA was described as counts per minute pernumber of cells per sample and expressed as “% normalizedratio of controls”. The uptake was calculated by subtractinguptake in treated cells from control (untreated) cells. Allexperiments were performed in triplicate and repeated twice.

2.2. Animal model of pheochromocytoma

All animal studies were conducted in accordance with theprinciples and procedures outlined in the National Institutes

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of Health Guide for the Care and Use of Animals andapproved by the Institutional Animal Care and UseCommittee. Animals were housed in a pathogen-free facilityunder a 12-hour day/night cycle and had ad libitum access tofood and water.

Our in vivo tumor model involved injections of MPC cells(suspended in 100 µl of sterile PBS) into two separate cohortsof 8–10 weeks old female athymic nude mice (NCr-nu,Taconic, Germantown, NY, USA). The first cohort of mice(n=9) received injection of 1×106 cells into the tail vein. Acontrol group of mice (n=5) received intravenous injection of100 µl PBS alone. The second cohort consisted of mice (n=4)that received s.c. injection of 5×106 cells. Mice wereobserved three times per week for evidence of distress,ascites, paralysis or excessive weight loss.

2.3. Anatomical imaging: microCT and MRI

Initial anatomical scans were conducted at 4 weeks postMPC implantation to determine the location and size ofmetastatic lesions. For both microCT and MRI, mice wereanesthetized with isoflurane during scanning. For microCT,mice were imaged using a MicroCAT-II scanner (Siemens,Knoxville, TN, USA) [23]. The hepatobiliary contrast agentFenestra Liver Contrast (LC) (Advanced Research Technol-ogies) was injected intravenously (13 µl of Fenestra LC pergram of body weight of animal), and microCT scans wereacquired 3 h post injection [24]. The utility of Fenestra LC inthis study has been described in detail previously [25,26].Reconstructions were performed using a cone-beam filteredback projection algorithm. The axial field of view (FOV)was set to 4.6 cm with an in-plane spatial resolution and slicethickness of 91 μm.

MRI was carried out using a 3 T MRI clinical scanner(Intera, Philips Medical System, Best, Netherlands) and adedicated 40-mm inner diameter solenoid coil (Intera). Forlongitudinal monitoring of lesions, a T2-weighted MRItechnique was chosen based on previously establishedparameters [24]. Mice were anesthetized with isofluraneand respiratory triggered T2-weighted images were acquiredwith the following parameters: FOV 8.0×8.0×2.0 cm3, datamatrix 512×512, 30–40 slices, TE/TR 65/4500 ms, flip angle90°, slice thickness 0.5 mm, 0.156×0.156 mm2 recon-structed resolution. No contrast agent was used for MRI.

Follow-up microCT or MRI scans were conducted againat week 5 after injection of MPC cells, wherein liver lesionswere approximately 3–5 mm in diameter and thus, suitablefor PET scanning. Anatomical imaging was not conductedon mice receiving cells s.c., as these tumors were detectedvisually and measured with digital calipers. Mice with s.c.tumors were imaged with PET approximately 2 weeks afterinjection of MPC cells or when tumor diameter reached 3–5mm. All nine animals with metastatic lesions were imagedusing all modalities, microCT, MRI and PET. [18F]-DOPAand [18F]-DA PET was performed in the same animals foruptake comparison and in all cohorts. [18F]-DA PET was

carried out within 24 h after MRI/CT imaging. [18F]-DOPAfollowed [18F]-DA PET imaging, also within 24 h.

2.4. Small animal PET imaging

Mice were anesthetized with isoflurane and received, viathe tail vein, injection of 100 μl of 3.4 to 4.3 MBq of [18F]-DA or 3.5–4.2 MBq of [18F]-DOPA. PET scanning wasperformed using the Advanced Technology LaboratoryAnimal Scanner (ATLAS) PET scanner [27], which has atransverse field-of-view of 6.8 cm and an axial FOV of 2cm. PET images were reconstructed by 2D-ordered-subsetexpectation maximization algorithm (five iterations and 16subsets), achieving a 1.5 mm full width at half maximumresolution at the center [28]. The reconstructed voxel sizewas 0.56×0.56×1.125 mm3. No correction was applied forattenuation or scatter. Mice were positioned in the proneposition on a heated animal bed near the center of FOV ofthe ATLAS scanner, where the highest image resolutionand sensitivity was available. Dynamic data acquisitions(10 min frame duration, up to six frames) were started about1 min after radiotracer injection and followed by wholebody scans (two to three bed positions, 10 min each) im-mediately after the dynamic acquisition. To detect possiblelung lesions, the whole body scans were set up to coverthe mouse body from the salivary glands to the urinarybladder. All acquisitions were recorded with a 100–700keV energy window.

2.5. Quantification of [18F]-DA and [18F]-DOPAaccumulation in organ and subcutaneouspheochromocytoma lesions

To assess the [18F]-DA and [18F]-DOPA radiopharma-ceuticals quantitatively, regions of interest (ROI) wereplaced over the location of the tumors and major organs(liver, kidneys, heart and muscles) in the dynamic and wholebody decay-corrected images. Radiotracer uptake in sampleswas reported as the maximum accumulation within tumors ororgans and was obtained from mean pixel values within themultiple ROIs. The results were calculated as standardizeduptake values (SUVs) [29]. We also used the maximaluptake within the tumor obtained from the most active voxelcluster located within the ROI. This is equivalent to themaximum standardized uptake value (SUVmax) used inclinical PET studies [30].

Quantitative analyses were carried out for all identifiedtumors and calculated as a liver tumor-to-background ratio(TBR) and a liver tumor-to-liver ratio (TLR). TBR is equal tothe SUVmax in tumors divided into SUVmax in muscle asnon-target structures (background). At the end of the study,an 18F-filled syringe with known activity concentration wasimaged to obtain the calibration factor, expressed in MBq of18F per counts per second (CPS), for the imaging system. AllROI values, given in CPS/ml, were multiplied by this factorand divided by injected activity per mouse body weight toyield SUV values. SUVmax in tumors was evaluated from

218 L. Martiniova et al. / Nuclear Medicine and Biology 39 (2012) 215–226

nine mice. The size of liver tumors was calculated from MRIas described previously [24]. Subcutaneous tumors weremeasured externally by calipers from week 2 onwards. Atweek 4, tumor size measurements were carried out. Lesionswere described as ‘small’ if less than 5 mm in diameter andas ‘big’ if larger than 5 mm.

No additional efforts were undertaken for the correctionof partial-volume effects and recovery. Three-dimensionalROI of tumors were determined using ATLAS software forthe subsequent data analysis. The maximum standardizeduptake value (SUVmax) was used to minimize errors due tothe partial volume effect [31].

2.6. Necropsy, histopathology andcatecholamine measurements

At the end of imaging, mice were sacrificed by cervicaldislocation. Tumor tissues and normal organs (from controlmice n=5) were removed and small pieces were snap frozenand stored at −80°C for catecholamine assays. Theremainder was stored in 10% formalin. Tumor catechol-amine levels were measured by high-pressure liquidchromatography after batch alumina extraction [32]. Thepresence of organ or s.c. pheochromocytoma lesions wasfurther characterized by histopathological examination.

2.7. Immunohistochemistry

Staining was performed on formalin-fixed paraffinembedded tissue sections obtained from lesions locatedacross multiple sites (liver, bone, ovary, adrenal glands).Sections stained for VMATs were processed according to

Table 1Comparison of tumors' localization by various imaging methods and confirmed w

Tumors' localization

Adrenal Ovary

MicroCT 0 0MRI 9 in 5 mice 4 in 4 mice[18F]-DA 8 in 4 mice 4 in 4 mice[18F]-DOPA 9 in 5 mice 4 in 4 miceNecropsy 18 in 9 mice 4 in 4 mice

Mouse name Necropsy and histopathology confirmation1 Multiple liver tumors (10, approx. 2–4 mm diameter); 2 e2 Multiple liver tumors (N10, ranging from 1–5 mm diam

in diameter.3 Single liver lesion (2.5 mm in diameter), enlarged ad4 Multiple liver tumors (N10, ranging from 1–4 mm di5 Multiple liver tumors (N20, ranging from 1–3 mm di6 Multiple liver tumors (N10, ranging from 1–5 mm diam

in diameter; 1 lung tumor, 2 mm in diameter.7 Multiple liver tumors (10, ranging from 1–3 mm diamete8 Multiple liver tumors (N5, ranging from 1–5 mm diam9 Multiple liver tumors (N20, ranging from 1–3 mm dia

diameter; approx. 20 lung tumors, 0.5 mm in diameteAll enlarged adrenal glands were confirmed as pheochromocytomas.

MicroCT, MRI and PET imaging were performed at Week 5. All imaging modaall nine mice.

previously described methods [33] using the primaryantibodies rabbit anti-VMAT1 (1:100; Chemicon, Temec-ula, CA, USA) and goat anti-VMAT2 (1:500; Santa CruzBiotechnology, Santa Cruz, CA, USA). Sections were alsostained for NET using a rabbit anti-NET primary antibody(1:50; kindly provided by Dr. Christie, University ofAuckland New Zealand [34]) and sheep anti-tyrosinehydroxylase (TH, 1:4000, kindly provided by Prof. P.Pilowsky, Macquarie University, Sydney Australia). ForNET/TH staining, methods were identical with theexception of the blocking solution, which contained 20%donkey and 20% rat serum (Jackson Immuno, Westgrove,PA, USA) in place of 10% donkey serum. For all sections,primary antibodies were detected with species-specificsecondary antibodies consisting of donkey anti-rabbitCy3, anti-goat FITC and anti-sheep DyLight. 488 (1:500;Jackson ImmunoResearch Laboratories, West Grove, PA,USA). Negative control sections were processed concur-rently according to the same methods, with omission of theprimary antibody. Sections were examined using a BioRadMRC-1024 confocal microscope at excitation wavelengthsof 488 and 568 nm and emission filters consisting of 522/35and 585LP, respectively. The measurement of importancein the review of immunohistochemical slides was thepresence of positive tumor cell staining. For each antibody,this was graded semiquantitatively on a five-tiered scalewhere 0=less than 10% of tumor cells positive; 1+=10–25%positive cells; 2+=25–50% positive cells, presented asmoderate staining; 3+=50–75% positive cells, moderate tostrong staining; and 4+=more than 75% of tumor cellspositive, strong staining [35].

ith necropsy in mice injected with MPC cells

Lung Liver Subcutaneous

0 0 NAN20 in 2 mice N 50 in 9 mice NA0 30-40 in 8 mice 01 in 1 mouse N50 in 9 mice 4 in 4 miceN20 in 2 mice N50 in 9 mice 4 in 4 mice

nlarged adrenal glands, 2.5 mm in diameter; 1 ovarian tumor, 3 mm in diameter.eter), 2 enlarged adrenal glands, 2.5 mm in diameter; 1 ovarian tumor, 4 mm

renal glands, 2 mm in diameter.ameter), enlarged adrenal glands, 2 mm in diameter.ameter), 1 adrenal gland tumor, 3 mm in diameter.eter), 2 adrenal gland tumors, 3 and 5 mm in diameter; 1 bone tumor, 5 mm

r), 1 ovarian tumor, 4 mm in diameter; 2 adrenal gland tumors, 3 mm in diameter.eter), 2 adrenal gland tumors, 3 mm in diameter.

meter); 1 ovarian tumor; 3 mm in diameter; 2 adrenal gland tumors; 3 mm inr.

lities followed by necropsy were performed within a 24 h time window in

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2.8. Statistical methods

Quantitative data for the in vitro and PET experimentswere presented as means±S.E.M. To determine whether thetwo radiotracers concentrations observed in the same tumorsvs. background tissues like liver and muscles differedsignificantly, we applied one-way analysis of variancefollowed by Neuman–Keuls test for group comparisons.Pb.05 was considered to be statistically significant.

3. Results

3.1. Anatomical imaging

Hepatic contrast agent allowed microCT-based visuali-zation of liver lesions as small as 0.35 mm in diameter,usually at the fourth week after tail vein injection of MPCcells. However, adrenal, lung and ovarian lesions remainedundetected by microCT in all animals, even at week 5(Table 1). Liver and lung lesions were localized using T2-weighted MRI acquisitions with respiratory triggering iflarger than 0.5 mm in diameter, usually by week 4. MRIdetected numerous tumors in the livers of all animals.Multiple tumors in lungs of two of the nine animals weredetected by MRI and confirmed at necropsy (Table 1).Enlarged adrenal glands and ovaries larger than 2.7 mm indiameter on MRI were considered as potential tumors as itwas closely described before [24]. The same adrenal glandtumors were visible by PET. However, those smaller than2.7 mm in diameter were also confirmed with microscopicpheochromocytomas. MRI detected additional four metas-tases in the ovaries of four of nine mice and ninemetastases in adrenal glands in five of nine mice. Necropsy

Fig. 1. Pharmacokinetics of uptake and retention of [18F]-DA (A) and [18F]radiopharmaceuticals in athymic nude mice. Each bar represents mean±S.E.M. valiver, the ideal time to acquire images for quantitative analysis of liver lesions was aversus uptake in the liver.

confirmed the evidence of tumors at these sites (Table 1).Outcome from microCT and MRI confirmed that sizes ofmetastases were suitable for PET imaging at week 5 afterMPC cells were injected.

3.2. Quantification of [18F]-DA and [18F]-DOPAaccumulation in organ and subcutaneouspheochromocytoma lesions

Dynamic PET scans were recorded for up to 70 minafter injection of both radiopharmaceuticals. Time–activitycurves with [18F]-DA (Fig. 1A) showed that the SUVmax

for liver lesions varied depending on a tumor size. Livertumors larger than 3 mm showed a decrease in SUVmax

over time, while lesions smaller than 3 mm remainedrelatively stable. In comparison, the SUVmax for [18F]-DOPA (Fig. 1B) in liver lesions remained stable over timeregardless of tumor size. [18F]-DA and [18F]-DOPAuptake in liver lesions smaller than 3 mm was notsignificantly different from the uptake in the liver up to 50min after their administration. Therefore, for both radio-pharmaceuticals, the optimal time for lesion detection was60–70 min after their administration, at which time TLRand SUVmax were the highest in both small and largerlesions, compared to liver background (Pb.001; Fig. 1Aand B). The TLR for liver lesions with [18F]-DA was1.83–2.83 depending on tumor size, whereas the TLR with[18F]-DOPA was 2.26–2.71 (Table 2).

Uptake of both radiopharmaceuticals by the kidneys andliver (Table 2) was observed in both control mice and tumorbearing mice, but a significantly higher uptake was clearlyapparent within metastatic lesions compared to normalunaffected tissue regions (representative coronal images

-DOPA (B) by liver tumors between 10–70 min after injection of thelues for nine mice. From comparisons with background radioactivity in thet 60–70 min after injection of [18F]-DA and [18F]-DOPA. ⁎Pb.05, ⁎⁎Pb.001

Table 2SUVmax in organs and tumors 60–70 min after administration of PETradioligands in mice harboring subcutaneous and organ metastaticpheochromocytomas

[18F]-DA [18F]-DOPA

SUVmax

(mean±S.E.M.)TLR TBR SUVmax

(mean±S.E.M.)TLR TBR

Kidney 2.41±0.45 – – 1.73±0.34 – –Muscle 0.31±0.02 – – 0.29±0.02 – –Liver 0.88±0.11 – – 1.33±0.14 – –Liver b3 mm 1.61±0.17 1.83 5.19 3.01±0.27 2.26 10.38Liver N3 mm 2.49±0.08 2.83 8.03 3.61±0.19 2.71 12.45Adrenal

N3 mm2.18±0.39 2.47 7.03 6.61±0.64 2.83 22.79

OvaryN3 mm

1.99±0.27 2.26 6.41 2.59±0.32 2.83 8.93

s.c. b5 mm 0.31±0.02 0.22 1.72 2.88±0.88 2.24 7.2s.c. N5 mm 0.35±0.12 0.27 1.94 2.00±0.06 1.56 5.0

A muscle uptake was taken as a background.

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shown in Fig. 2 and longitudinal images in Fig. 3). There wasadditional uptake apparent for [18F]-DOPA in stomach wall,pancreas and spleen. Liver lesions imaged once a week fortwo weeks, showed a time dependent lowering of [18F]-DAuptake (the first week 1.85±0.199 SUVmax; the second week1.16±0.07 SUVmax; Pb.05), whereas uptake of [

18F]-DOPA

Fig. 2. Combined coronal MRI images of representative mouse superimposed withanimal (A) and an animal previously injected with MPC cells (B). The color scale iliver tumors with [18F]-DA is 1.5–2.5 SUVmax and for [18F]-DOPA it is 3–3.6 SU

revealed similar uptake over the same time period (the firstweek 3.023±0.22 SUVmax; the second week 2.88±0.12SUVmax; not significant).

Uptake of [18F]-DA by s.c. tumors differed significantlyfrom that observed in organ lesions (Fig. 3G). [18F]-DAshowed very low accumulation in both smaller and larger s.c.tumors (0.31±0.02 SUVmax for tumors b5 mm and 0.35±0.12SUVmax for tumors N5 mm), almost at the level ofbackground within muscle (0.31±0.02 SUVmax,). However,the uptake of [18F]-DOPA within the same tumors was muchhigher [2.88±0.88 SUVmax for tumors b5 mm and 2.0±0.06SUVmax for tumors N5 mm, (Pb.001) than the uptake for[18F]-DA (TLR=1.56–2.24 vs. 0.22–0.27; Table 2)]. Forboth the organ metastatic and s.c. tumor cohorts, the uptakeobserved in major organs and tumors was correlated withbiodistribution studies (data not presented).

Thirty to forty liver lesions were localized using [18F]-DA PET in eight out of nine animals, while [18F]-DOPAPET localized more than 50 liver lesions in nine animals(Table 1). Eight adrenal lesions were detected with [18F]-DA in four out of nine animals compared to nine detectedin five out of nine with [18F]-DOPA. Four ovarian lesionswere visible in four of nine animals with bothradioligands, and single lung lesion was only visualizedwith [18F]-DOPA.

PET images after administration of [18F]-DA and [18F]-DOPA to a controlndicates maximal standardized uptake values (SUVmax). The usual range forVmax.

Fig. 3. Localization of pheochromocytoma lesions by [18F]-DA or [18F]-DOPA PET in representative animals after i.v. (Panels 1–E) and s.c. (Panel G)injections of MPC cells. Upper panels, A and B, show microCT coronal images using Fenestra contrast agent with respiratory gating in a representativeanimal after i.v. injection of MPC cells. The animal was imaged on days 38 through 47 after i.v. injection of MPC cells, where panels C and E show rapidgrowth of liver lesions visible using [18F]-DA PET over a 7-day time period. [18F]-DOPA PET visualization of the same animal over consequent days isshown in panels D and F. Images were acquired at 60–70 min after injection of radiopharmaceuticals. The localization of a large s.c. tumor depicted bytransverse and coronal images after s.c. injection of [18F]-DA and [18F]-DOPA into another representative animal is shown in Panel G. [18F]-DA PETimaging reveals very low accumulation of the radiopharmaceutical in the tumor (0.35 SUVmax), almost at the level of nontarget tissue such as muscle (0.31SUVmax). In contrast, the uptake of [18F]-DOPA by the same tumor was much higher (2 SUVmax). Circles identify the tumor on both transverse and coronalimages and arrows points to the liver.

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3.3. Assays of [18F]-DA and [18F]-DOPA uptake andretention in vitro

Desipramine had a stronger inhibiting effect compared toreserpine on accumulation of [18F]-DA during the initial 10-min uptake phase (Pb.05; Fig. 4A). This indicates a strongdependence of NET for [18F]-DA uptake. Desipraminedecreased [18F]-DA uptake to 14.3% relative to untreatedcells (Pb.001), whereas reserpine decreased [18F]-DAuptake to 18.7% (Pb.001) and tetrabenazine 20.07%(Pb.001). The combination of desipramine and reserpinefurther decreased [18F]-DA uptake to 2.75% (Pb.0001). Incontrast, reserpine had a stronger inhibiting effect thandesipramine on the accumulation of [18F]-DA after the 120min retention phase of incubations (Pb.001; Fig. 4B).Accumulation of [18F]-DA relative to control was decreased(Pb.001) to 40.15% after desipramine, 21.45% afterreserpine, 33.83% after tetrabenazine and 25.85% aftercombination of desipramine and reserpine.

Relative to control cells, uptake of [18F]-DOPA after the10-min incubation period was decreased (Pb.001) to 67.37%after desipramine, 14.03% after reserpine, 17.87% aftertetrabenazine and 13.47% after the combination of desipra-mine and reserpine (Fig. 4C). In contrast, after the 120-min

incubation period, there was no significant difference inretention of [18F]-DOPA between desipramine treated andcontrol cells (Fig. 4D). The uptake of [18F]-DOPA was,however, decreased (Pb.001) to 11.8% of control valuesafter reserpine, to 12.1% after tetrabenazine (Pb.001) and to7.97% after the combination of desipramine and reserpine(Pb.001). These results indicate strong importance ofVMAT2 in accumulation and retention of [18F]-DOPA inMPC cells. Statistical differences between treatments withreserpine, tetrabenazine and combination of desipramineand reserpine, compared to desipramine alone, are presentedin Fig. 4.

3.4. Histopathology and immunohistochemistry: expressionof monoamine transporters and tyrosine hydroxylase

Histopathology confirmed all lesions to be pheochromo-cytomas. Furthermore, all lesions contained high levels ofcatecholamines, relative to normal tissues (Table 3). Thelowest ranges for expression of NE were found in s.c.lesions. All analyzed samples (2× adrenal gland lesions, 1×bone lesion, 3× liver lesions, 1× lung lesions, 1× ovarianlesion, 3 s.c. lesions) were positive for TH staining (Table 4),ranging from 2+–4+ positive staining on a five-tier scale.

Fig. 4. Uptake of [18F]-DA (A and B) and [18F]-DOPA (C and D) in MPC cells presented as “normalized ratio of controls” at 10 and 120 min after treatment with1 μM desipramine (DMI), 10 μM reserpine (RES), 10 μM tetrabenazine (TET) and their combination (30 min before adding the [18F]-DA and [18F]-DOPA).⁎Pb.05, ⁎⁎Pb.001 ⁎⁎⁎Pb.0001 versus control (“C”). +Pb.05, ++Pb.001 versus desipramine (“DMI”).

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Ten of 11 samples showed NET staining (1+–4+).Expression of VMAT was more variable, 9 of 11 samplesshowing VMAT2 (2+-4+) and only 4 of 11 samples showingVMAT1 expression (1+). Furthermore, when VMAT1 wasexpressed (Fig. 5), it was only present in isolated clusters ofcells throughout the section, in contrast to the other markerswhere expression was generally more widespread in theirdistribution pattern. Interestingly, all s.c. lesions expressedVMAT2 but lacked expression of VMAT1. All markerswere localized to the cytoplasm.

4. Discussion

The present study compared metastatic and s.c. pheo-chromocytoma models and established differences in theutility of PET imaging with [18F]-DA and [18F]-DOPA andanatomical imaging with microCT and MRI. While [18F]-

DA and [18F]-DOPA PET performed equally well for thedetection of ovarian metastatic tumors, [18F]-DOPA PETshowed superiority to [18F]-DA PET in the detection of liver,lung and s.c. tumors. In particular, s.c. tumors were detectedonly with [18F]-DOPA PET. Furthermore, comparisons ofthe in vitro uptake of both radiocompounds by MPC cellsconfirmed the importance of the NET for uptake of [18F]-DAand also confirmed that VMAT2 and, to a lesser extent,VMAT1 is important for its retention in catecholaminestorage vesicles. The VMAT2 role for the [18F]-DA entryinto catecholamine storage vesicles was also well demon-strated from [18F]-DOPA results. It is known that [18F]-DOPA is converted to [18F]-DA in cytoplasm and indeed ourexperiments using tetrabenazine and [18F]-DOPA showedthe most diminished uptake of [18F]-DOPA, actually [18F]-DA and its storage and retention in catecholamine vesicles.

As supported by several clinical studies [36–38]functional imaging using [18F]-DA and [18F]-DOPA PET

Table 3Tissue catecholamine concentrations (pg per mg of wet weight tissue) inMPC cell-derived tumors compared to concentrations in normal tissues ofcontrol female nude mice that did not receive injections of MPC cells

Catecholamines (pg/mg)

NE EPI DA

Normal valuesLiver 25–125 9–123 –Adrenalglands

14,548–208,181 27,611–333,150 –

Ovaries 189–411 – –Lung 129–256 54–254 –

Tumor valuesLiver tumors 80,707–1,225,792 6496–24,716 19,532–96,128Adrenaltumors

262,415–454,471 442,572–758,463 9325–95,300

Ovariantumors

62,495–414,566 – 31,301–65,232

Lung tumors 95,431–4,117,381 – 29,184–1,907,137Subcutaneoustumors

95,098–234,637 – 37,314–54,085

Catecholamine concentrations are shown as ranges of values. EPI,epinephrine; DA, dopamine.

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is a useful tool for diagnostic localization of pheochromo-cytomas and paragangliomas (extra-adrenal pheochromocy-tomas). Surgical resection of metastatic lesions is usuallyimpossible, and biopsies are dangerous due to profoundcatecholamine release and a possibility of hypertensive crisisand lethal tachyarrhythmia. Our metastatic animal modelprovides a valuable resource for a better understanding oftumor characteristics related to experimental imaging andfuture targeted therapeutic interventions. We found a visiblylower accumulation of [18F]-DA compared to [18F]-DOPAin large liver lesions, presumably due to the decrease in the

Table 4Expression of TH, NET, and VMATs in tumor samples (0=less than 10% oftumor cells positive; 1+=10–25% positive cells; 2+=25–50% positive cells,presented as moderate staining; 3+=50–75% positive cells, moderate tostrong staining; and 4+=more than 75% of tumor cells positive, strongstaining)

Location TH NET VMAT1 VMAT2 [18F]-DA [18F]-DOPA(SUVmax)

Adrenal 1 4+ 2+ 1+ 4+ 2.15 6.46Adrenal 2 4+ 3+ 1+ 3+ 1.98 5.79Bone 4+ 2+ 0 0 — 1.42Liver 1 3+ 1+ 0 3+ 2.46 3.31Liver 2 4+ 3+ 1+ 4+ 1.89 2.79Liver 3 2+ 0 0 0 — 3.24Lung 2+ 4+ 0 3+ — 1.15Ovary 4+ 2+ 1+ 2+ 1.92 3.05s.c. 1 4+ 2+ 0 2+ — 1.88s.c. 2 3+ 3+ 0 3+ — 2.25s.c. 3 4+ 4+ 0 2+ — 2.09

[18F]-DA and [18F]-DOPA PET uptake in organ metastatic and s.c. tumorsexpressed in SUVmax; calculated from images after 60–70 min afteradministration. Numbers 1–3 indicate number of samples. A dash sign “—”presents SUVmax values less than 0.3 for both [18F]-DA and [18F]-DOPA.

NET expression secondary to tumor dedifferentiation. [18F]-DA uptake in liver lesions also decreased over longitudinalimaging, whereas for [18F]-DOPA there was no decrease inthe same tumors imaged over time. The differences in uptakeof these two radiopharmaceuticals may be relevant to recentclinical findings of differences in functional imagingcharacteristics of various pheochromocytomas dependingon their underlying mutations and nature [38,39]. Forexample, in patients with malignant paragangliomas due tosuccinate dehydrogenase subunit B mutations, [18F]-DA issuperior to [18F]-DOPA for localization of metastases(reverse to the present animal model of pheochromocytoma),whereas in other patients with so called ‘head-and-neck’paragangliomas, [18F]-DOPA is superior to [18F]-DA [37].These tumor differences in PET imaging characteristics aresuggested to reflect variations in the expression of NET,VMATs and perhaps amino acid transporter and are crucialin a current view of the choice of pheochromocytomaimaging modality.

Comparing the uptake of both radiopharmaceuticals ins.c. tumors revealed also differences. For example, s.c.tumors showed a high uptake of [18F]-DOPA but no uptakeof [18F]-DA above the level of background. Threepossibilities could explain these findings (1) differences ofblood delivery of [18F]-DA and [18F]-DOPA into organmetastatic and s.c. tumors; (2) the expression of cellmembrane, vesicular, and amino acid transporters and theirfunction and (3) the conversion of [18F]-DOPA to [18F]-DAin tumor cytoplasm and their intracellular storage orretention mechanisms. The half-life of dopamine in circula-tion is about 1 minute, and the half-life of L-DOPA is about40–60 min [40]. If s.c. tumors would not have enough bloodsupply comparing to organ metastases, taking into aconsideration a different half-life of dopamine and L-DOPA in plasma, this could be an important componentfor a lower uptake of [18F]-DA to [18F]-DOPA in thesetumors. Another possible explanation would be the expres-sion and function of NET. Even though the expression ofNET was present in s.c. tumors and organ metastases, itsfunction could be diminished to a greater extent in s.c. thanin organ lesions. In the present animal model, the expressionof NET and VMATs among excised organ and s.c. tumorswas highly variable, a finding similar to previous resultsabout NET expression in various human pheochromocytomatissue samples based on their genetic background, locationand perhaps metastatic potential [33]. Even though clustersof the NET showed different patterns of expressionthroughout organ and s.c. tumor samples, there was nodistinct feature that could clearly explain the different [18F]-DOPA and [18F]-DA PET results for these lesions based onthe presence of the NET. Interestingly, catecholaminemeasurements revealed lower values of norepinephrine(NE) in s.c. tumors comparing to organ metastases.Furthermore, VMAT1 exhibited limited expression com-pared to VMAT2, particularly in s.c. tumors when comparedwith organ metastatic lesions. In normal adrenal chromaffin

Fig. 5. Expression profile of NET, TH and VMATs in s.c. (A–D) and liver (E–H) lesions. While the expression of TH (B, F) was consistently detected, NET (A,E) expression was variable, with strong immunoreactivity in some samples (A) but more diffuse staining with interspersed foci of strong immunoreactivity (E;arrows) in others. Typically, VMAT1 (C, G) expression was absent (C), but in some samples (G) isolated clusters of highly immunoreactive cells (arrows) weredetected. Diffuse VMAT2 (D, H) staining was typically detected, including some highly immunoreactive clusters of cells (H; arrows). Bar scale: (A, B, E and F)100 μm, (C, D, G and H) 50 μm.

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and enterochromaffin cell, VMAT1 is the predominantisoform, whereas VMAT2 is typically the predominantisoform expressed in neurons of central autonomic nervoussystem [21,41]. This is well documented from previousstudies where the uptake of [18F]-DOPA in the centralnervous dopaminergic system depended on the presenceVMAT2 [42,43]. The present results suggest that VMAT2 ismore important that VMAT1 in the accumulation of [18F]-DA and [18F]-DOPA (converted in cytoplasm to [18F]-DA)in both organ metastatic and s.c. tumors.

There are several limitations to the present study. First,the proper function of the VMAT1 has not been investigated.Second, we did not study any presence and function of thecell membrane amino acid transporter system that is knownto allow L-DOPA to enter a pheochromocytoma cell; lastly,the absence of in vivo experiments manipulating (e.g.,inhibiting) NET, VMAT1 and VMAT2, and amino acidtransporters. However, in our preliminary experiments, theadministration of desipramine, reserpine and tetrabenazine inpheochromocytoma animal model with extensive liver andother organ lesions was difficult, and side effects were inmost cases fatal.

5. Conclusion

In summary, this is the first study to compare multi-imaging modalities with [18F]-DOPA and [18F]-DA PET,microCT and MRI for detection of organ metastatic and s.c.

lesions in nude mice model of pheochromocytoma. Thepresent data has potential relevance for explaining differ-ences in imaging characteristics in patients with variouspheochromocytomas and perhaps other neuroendocrinetumors. Subcutaneous xenografts, as in vivo tumor model,remains the most popular method for testing new imaging ortreatment approaches [44]. Our data suggests that theradiotracer tumor uptake in pheochromocytomas is differentwhen using s.c. models, as opposed to organ metastaticmodels, to study the efficacy of new imaging agents.

The data should also prove useful to other studies utilizinganimal models of metastatic disease in which it is desirable,if not essential, to obtain longitudinal information about thetumor development and tumor responses to treatment.

Acknowledgments

We would like to acknowledge the valuable assistance ofthe colleagues in our group; we thank Mr. Stephen Uyenoand Mr. Eli Thompson for their assistance with animals. Thisresearch was supported (in part) by the Intramural ResearchProgram of the Eunice Kennedy Shriver National Institutesof Child Health and Human Development, Warren GrantMagnuson Clinical Center, National Institute of Neurolog-ical Disorders and Stroke, National Institute of BiomedicalImaging and Bioengineering, National Cancer Institute, bythe grant from the PheoPara Alliance (to J.P.) and APVV-0148-06 (to R.K.). The authors have no conflict of interestto disclose.

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